1. Field of the Invention
This invention relates generally to methods used for performing combinatorial chemistry and more particularly to a method and apparatus for the parallel synthesis of chemical arrays using spatially addressable photochemical reaction schemes.
2. Description of the Related Art
Currently there is widespread interest in using combinatorial libraries of oligonucleotides, polypeptides, synthetic oligomers and small organic molecules to search for biologically active compounds (Kramer, et al., 1993; Houghten, et al., 1992, 1991; Dooley, et al., 1993a-1993b; Eichler, et al., 1993; Pinilla, et al., 1992, 1993). For example, ligands discovered by screening libraries of these types may be useful in mimicking or blocking natural ligands, or interfering with the naturally occurring interactions of a biological target. They can also provide a starting point for developing related molecules with more desirable properties, e.g., higher binding affinity.
Combinatorial libraries of the types useful in this general application have been formed by various solid-phase or solution-phase synthetic methods. In one approach, beads containing successive precursors to the target compounds that form the library are alternately mixed and separated, with one of a selected number of reagents being added to each group of separated beads at each step (the “split-mix” method: Furka, et al., 1991; Chen et al., 1994; Pham, et al., 1995; Dillard, et al., 1994). Each bead contains only one chemical species, allowing the beads themselves to be used for screening. However, the identity of the species on each bead must be independently determined. Although several methods have been reported for tagging the support beads with molecules more readily analyzable than the library members themselves (e.g., Nestler, et al., 1994; Felder, et al., 1995; Dillard, et al., 1994), the need for separate identification of each species nonetheless limits the usefulness of this approach for the preparation of very large libraries.
Another general approach involves the synthesis of a combinatorial library as a physically segregated array of compounds (Geysen, et al., 1984, 1985; Southern, 1994; Southern, et al., 1992; Bunin, et al., 1992, 1994; DeWitt, et al., 1993). Libraries of compounds have been synthesized on functionalized resins either coated on (Geysen, et al., 1984, 1985; Bunin, et al., 1992, 1994) or contained within (DeWitt, et al., 1993) arrays of pins, with reactions carried out in separate chambers. Using such an approach, the chemical identity of each library element on the array is associated with an addressable position on the array. However, in this method, as with the split-mix method, preparation of large libraries would require an undesirably high number of manipulations and/or a large array of separate reaction vessels or sites.
A method for preparation of potentially high density position-addressable arrays on a planar substrate has been reported (Fodor, et al., 1991; Pirrung, et al., 1992). In this method, applicable primarily to oligomeric compounds, a substrate having photoprotective groups is irradiated, using photolithographic mask techniques, in selected regions only, to deprotect surface active groups in those selected regions. The entire surface is then treated with a solution of a selected subunit, which itself has a photoprotected group, to react this subunit with the surface groups in the photodeprotected regions. This process is repeated to (i) add a selected subunit at each region of the surface, and (ii) build up different-sequence oligomers at known, addressable regions of the surface. This method allows for the synthesis of very large permutation libraries, e.g., 104-106 compounds, in a position addressable array by parallel subunit addition. For example, in the case of oligonucleotide libraries, each subunit addition step requires only four addition reactions, one for each nucleotide added. Thus, in the production of a library of oligonucleotide compounds where n=8, i.e., a library of 8-mers, 65,536 oligonucleotide compounds can be constructed with a total of 32 reaction steps (8 subunit additions, 4 reactions each).
In cases where the compounds are to be screened for biological activity while, still attached to the substrate, this method also allows for rapid screening by binding a reporter-labeled target to the surface and determining the positions of bound target. Surface arrays of this type may be used both for combinatorial library screening (Fodor, et al., 1995; Geysen, et al., 1984, 1985) or for various types of oligonucleotide analysis, such as sequencing by hybridization (Drmanac, et al., 1993; Southern, 1994; Southern, et al., 1992). However, such planar arrays are necessarily limited in the amount (number of molecules) of each library species, since the planar region available to each species is quite small, e.g., on the order of 102-103 μm2. As a consequence, the ability to detect binding species on the array may be limited. Further, it is not feasible to carry out solution-phase screening on a planar array, because of the difficulty of physically separating different array regions carrying different library members.
It would thus be desirable to provide a method and apparatus for preparing a large combinatorial library of compounds which has the advantages of (i) parallel synthesis of subunits in known, addressable library positions, (ii) adaptable to virtually any oligomer or small-molecule chemistry, (iii) a relatively large area for synthesis of each library member, and (iv) screening of individual library compounds in either solution phase or solid phase. The present invention is directed to meeting such objectives, and in doing so, overcoming, or at least reducing the effects of, one or more of the problems set forth above.